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. 1999 Jan 1;13(1):49-63.
doi: 10.1101/gad.13.1.49.

Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB

J A Ranish  1 , N Yudkovsky, S Hahn
Affiliations

Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB

J A Ranish et al. Genes Dev. .

Abstract

Assembly and activity of yeast RNA polymerase II (Pol II) preinitiation complexes (PIC) was investigated with an immobilized promoter assay and extracts made from wild-type cells and from cells containing conditional mutations in components of the Pol II machinery. We describe the following findings: (1) In one step, TFIID and TFIIA assemble at the promoter independently of holoenzyme. In another step, holoenzyme is recruited to the promoter. Mutations in the CTD of Pol II, Srb2, Srb4, and Srb5, and two mutations in TFIIB disrupt recruitment of all holoenzyme components tested without affecting TFIID and TFIIA recruitment. These results indicate that the stepwise assembly pathway is blocked after TFIID/TFIIA binding. (2) Both the Gal4-AH and Gal4-VP16 activators stimulate formation of active PICs by increasing the extent of PIC formation. The Gal4-AH activator stimulated PIC formation by enhancing the binding of TFIID and TFIIA, whereas Gal4-VP16 could enhance the recruitment of TFIID, TFIIA, and holoenzyme. (3) Extracts deficient in TFIIA activity showed reduced assembly of all PIC components. These and other results suggest that TFIIA acts at an early step by enhancing the stable recruitment of TFIID. (4) An extract containing the TFIIB mutant E62G, had no defect in PIC formation, but had a severe defect in transcription. Similarly, mutation of the TATA box reduced PIC formation only two- to fourfold, but severely compromised transcription. These results demonstate an involvement of TFIIB and the TATA box in one or more steps after recruitment of factors to the promoter.

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Figures

Figure 1
Figure 1
Immobilized templates used in this study. HIS4 consists of the HIS4 core promoter containing the TATA box and start sites of transcription, with one Gal4 site positioned upstream of the TATA box. The Pst1 site was used for cleavage of the promoter from the beads after formation of complexes. (x) Approximate location of bases that were changed to eliminate cryptic TATA sequences. HIS4mTATA is identical to HIS4 except the TATA box is replaced with a GC-rich sequence. Promoterless lacks the HIS4 promoter but retains the Gal4 site and upstream DNA.
Figure 2
Figure 2
Promoter-dependent PIC assembly and activity. (a) PIC assembly on the HIS4, HIS4mTATA, and Promoterless templates. Nuclear extract was incubated with the indicated immobilized templates for 40 min with or without Gal4–AH or Gal4–VP16. PICs were isolated and analyzed as described in Methods and Materials. (Lane 1) Dynabeads lacking DNA were incubated with Gal4–AH and nuclear extract. (b) Transcription activity of PICs formed on the HIS4-immobilized template. PICs were formed as described in a. For multiround measurements, PICs were incubated with NTPs for 60 min (lanes 1–3). Single-round measurements were obtained by incubating PICs with NTPs for 2 min (lanes 4–6). Activity of washed complexes was measured by resuspending PICs in transcription buffer containing NTPs for 10 min (lanes 7–9). Start sites were mapped by primer extension. (Brackets) α-amanitin sensitive start sites. (c,d) Transcription activity of PICs formed on the HIS4 and HIS4mTATA promoter templates. (c) PICs were formed as described in a on the HIS4 (lanes 1-3) and HIS4mTATA (lanes 4–6) immobilzed templates, and then incubated with NTPs for 40 min. Start sites were mapped by primer extension. (d) PICs were formed on the HIS4 (lanes 1 and 3) and HIS4mTATA (lane 2) promoter templates (pSH515 and pSH514) by incubation with nuclear extract and Gal4–AH. Single-round measurements were obtained by incubating PICs with NTPs for 2 min. RNAs were detected by S1 nuclease protection assay. α-Amanitin (α-a)-sensitive bands used for quantitation are indicated by brackets. (*) Position of the undigested probe.
Figure 3
Figure 3
An extract containing a TBP mutant is defective in stable PIC formation and activity. (a) Transcription activity of PICs formed in wild-type and TBP(I143N) extracts. Experimental design is outlined at top. The indicated extracts were incubated with the HIS4 immobilized template and Gal4–AH for 40 min. rTBP (200 ng) was included where indicated. For multiround measurements, PICs were incubated with NTPs for 40 min (lanes 1,4,5). Single round measurements were obtained by incubating PICs with NTPs for 2 min (lanes 2,6,7). Activity of washed complexes was measured by resuspending PICs in transcription buffer containing NTPs for 2 min (lanes 3,8,9). (Brackets) α-Amanitin sensitive start sites. (b) PIC assembly on the HIS4 template in wild-type and TBP(I143N) extracts. PICs were formed as described in a except that reactions were scaled up twofold. PICs were analyzed as described in Fig. 2a.
Figure 4
Figure 4
Extracts containing mutant subunits of TFIIA are defective in stable PIC formation and activity. (a) Transcription activity of PICs formed in wild-type and TFIIA mutant extracts. Experimental design is outlined at the top. The indicated extracts were incubated with the HIS4 immobilized template and Gal4–AH for 40 min. rTFIIA (80 ng) was included where indicated. For multiround measurements, PICs were incubated with NTPs for 40 min (lanes 1,4,5,10,11). Single round measurements were obtained by incubating PICs with NTPs for 2 min (lanes 2,6,7,12,13). Activity of washed complexes was measured by resuspending PICs in transcription buffer containing NTPs for 2 min (lanes 3,8,9,14,15). Brackets indicate the α-amanitin sensitive start sites. (b) PIC assembly on the HIS4 template in wild-type and TFIIA mutant extracts. PICs were formed as described in a except that reactions were scaled up twofold. PICs were analyzed as described in Fig. 2a.
Figure 5
Figure 5
Mechanism of TFIIA action. (a) Rate of formation of active PICs in a wild-type and a Toa1-25 mutant extract. Experimental design is outlined at top. The indicated nuclear extracts were incubated with the HIS4 template (pSH515) and Gal4–AH, in the presence or absence of rTFIIA (40 ng). At the indicated time, an aliquot of the reaction was removed and added to a tube containing NTPs. After 2 min the reaction was stopped and analyzed by primer extension. (Brackets) α-Amanitin- sensitive start sites. (b) Graph of the quantitated data from a. (c) Stability of PICs to challenge by a second template in the presence and absence of TFIIA. Experimental design is outlined at top. Toa1-25 mutant extract was incubated with the HIS4 template (pSH515) and Gal4–AH for 40 min. rTFIIA (40 ng) was included in reactions 6–10. PICs were challenged by addition of a 20-fold molar excess of a HIS4 promoter template (pSH388) for either 10 min (lanes 3,8) or 60 min (lanes 4,9). Transcripts produced from this template are not detected with the primer used in these experiments. Single-round transcription was measured by addition of NTPs for 2 min. (Lanes 1,6) Single-round transcription of PICs formed for 40 min with no challenge, in the absence and presence of rTFIIA. (Lanes 2,7) Identical to lanes 1 and 6 except that pSH388 was added at the start of the incubations. (Lanes 5,10) Single-round transcription of PICs that have incubated for the same time as reactions 4 and 9 without pSH388.
Figure 6
Figure 6
Analysis of TFIIB mutant extract in PIC formation and activity. (a) Transcription activity of PICs formed in TFIIB mutant extracts. The indicated extracts were incubated with the HIS4 promoter template (pSH515) and Gal4–AH for 35 min. rTFIIB (30 ng) was included where indicated. After addition of NTPs, single-round transcription was measured by two methods: (1) sarkosyl was added to 0.2% after 45 sec (lanes 1,3) or 1 minute (lanes 5,7,9,11), followed by SDS stop buffer after 10 min; or (2) SDS stop buffer was added after 2.5 min (lanes 2,4,6,8,10,12). (Brackets) α-Amanitin-sensitive start sites. (b) PIC formation in TFIIB mutant extracts. PICs were formed by incubation of the indicated extracts with the HIS4 immobilized template and Gal4–AH for 40 min. rTFIIB (120 ng) was included in the incubation where indicated. PICs were analyzed as described in Fig. 2a.
Figure 7
Figure 7
The role of the CTD of Pol II in PIC formation and activity. (a) Transcription activity of PICs formed in a wild-type and a CTD9 extract. Experimental design is outlined at top. The indicated extracts were incubated with the HIS4 immobilized template and Gal4–AH for 40 min. For multi-round measurements, PICs were incubated with NTPs for 40 min (lanes 1,4). Single-round measurements were obtained by incubating PICs with NTPs for 2 min (lanes 2,5). Activity of washed complexes was measured by resuspending PICs in transcription buffer containing NTPs for 2 min (lanes 3,6). (Brackets) α-Amanitin-sensitive start sites. (b) PIC assembly on the HIS4 immobilized template in wild-type and CTD9 extracts. PICs were formed as described in a except that reactions were scaled up twofold. PICs were analyzed as described in Fig. 2a.
Figure 8
Figure 8
The role of Srb2, Srb4, and Srb5 in active PIC formation. (a) Transcription activity of PICs formed in a wild-type extract, and in Srb2, Srb4, and Srb5 mutant extracts. The indicated extracts were incubated with the HIS4- immobilized template and Gal4–AH for 40 min. rSrb2 (100 ng) was added to reactions 7–9. For multiround measurements, PICs were incubated with NTPs for 40 min (lanes 3,6,9,12,15). Single-round measurements were obtained by incubating PICs with NTPs for 2 min (lanes 2,5,8,11, and 14). Activity of washed complexes was measured by resuspending PICs in transcription buffer containing NTPs for 5 min (lanes 1,4,7,10,13). (Brackets) α-Amanitin-sensitive start sites. (b) PIC assembly on the HIS4 (W) and HIS4mTATA (M) immobilized templates in a wild-type extract and a Srb2 mutant extract. PICs were formed as described in a except that reactions were scaled up twofold. rSrb2 was included in reactions 5 and 6. PICs were analyzed as described in Fig. 2a. (c) PIC assembly on the HIS4-immobilized template in a wild-type extract and in Srb4 and 5 mutant extracts. PICs were formed as described in a except that reactions were scaled up twofold. PICs were analyzed as described in Fig. 2a.
Figure 9
Figure 9
Mechanism of PIC assembly. In the model, TFIID and TFIIA are stably recruited to the promoter by an activator. In the presence of Gal4–AH, the D–A-promoter complex is required for the stable recruitment of the Srb/Mediator holoenzyme. The strong activator Gal4–VP16 can recruit holoenzyme independently of the D–A-promoter complex. Importantly, after D–A recruitment, the stepwise assembly pathway is blocked. Alternative PIC assembly mechanisms are possible, including a concerted reaction or holoenzyme independent PIC formation depending on the promoter and activators involved. A postrecruitment role for the TATA box and TFIIB, such as open complex formation or promoter clearance, is indicated.
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